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Medical Microbiology. Disinfection and Sterilization. —— For the course of Medical Microbiology for MBBS foreign students, Class 2006/2011, SYSU September 18, 2007 Mengfeng Li ( 黎孟枫) , M.D. Department of Microbiology, Zhongshan School of Medicine, SYSU, Guangzhou, China

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Medical Microbiology

Disinfection and Sterilization

——For the course of Medical Microbiology for MBBS foreign students, Class 2006/2011, SYSU

September 18, 2007

Mengfeng Li (黎孟枫), M.D.

Department of Microbiology, Zhongshan School of Medicine, SYSU, Guangzhou, China

[email protected]


Early needs and success for disinfection (Early 1800’s) —— a historic story

  • By mid-1800’s, almost half of post-operative patients died of sepsis (then called “hospital disease”). A common report by surgeons was: operation successful but patient died.

  • A hypothesis: ① exposing moist body tissue to oxygen  sepsis;② best prevention: keeping air away from wounds by means of plasters, collodion or resins.

  • Having tried methods to encourage clean healing with no success, surgeon Joseph Lister discarded the concept of direct infection by bad air but postulated that sepsis might be caused by a 'pollen-like dust‘, although he did not know yet the “dust” was living microbes.

  • When Louis Pasteur suggested the presence of living organisms in the air, Lister made the connection with wound sepsis: the microbes in the air were likely causing the sepsis and should be destroyed before they entered the wound.

  • Lister had previously heard that 'carbolic acid' was used to treat sewage, and that fields treated with the affluent were freed of a parasite causing disease in cattle. He then began to clean wounds and dress them with carbolic acid..


The milestone:

case of James Greenlees

-Aug. 12, 1865, James Greenlees, 11-year-old

-Struck by a horse-drown cart

-Compound fractures

-Taken to Royal Infirmary

-Under the care of Joseph Lister

Joseph Lister operated with carbolic acid (石炭酸)spray


Why disinfection and sterilization?

  • Contagious diseases

  • Hospital infection (e.g., OR, ID ward) or other opportunistic infection

  • Lab contamination

  • Etc.

  • Microbes:

  • usually easy to grow in environment;

  • but also can be inhibited or killed by certain environmental (physical or chemical) factors/conditions.


Terminology

  • Disinfection消毒

  • Sterilization灭菌

  • Bacteriostasis抑菌

  • Antisepsis防腐

  • Asepsis 无菌


Disinfection(消毒)

  • Process of reducing or eliminating living pathogenic microorganisms in or on materials, so they are no longer a health hazard.

    For example: use of alcohol before drug injection.


Sterilization(灭菌)

  • Process of destroying all microbial forms. A sterile object is one free of all microbial forms, including bacterial spores.

  • More thorough than disinfection


Bacteriostasis(抑菌)

  • Process of inhibiting the growth of microorganisms, in vivo (mostly) or in vitro.

    For example: bacteriostatic antibiotics


Antisepsis (防腐)

  • Process of inhibiting or preventing growth of microbes, mostly in vitro and not bactericidal or sporicidal (杀芽胞的 )

    For example: use of chemical agents on skin, other living tissues or food/beverage.


Asepsis(无菌)

  • A state where no living microorganism exists.

    For example: OR (Operating Room)


Controlling Microorganisms with Physical Conditions

  • High Temperature (heat)

  • Radiation

  • Ultrasound

  • Filtration

  • Low Temperature

  • Desiccation(干燥)


static action

cidal action

High Temperature ——Dry heat and Moist heat  protein denaturation and clotting; DNA strand breakdown

.


Dry heat:protein oxidation

  • Incineration(焚烧)

    • most thorough (>500℃)

    • disposals and corpes

  • Flaming (burner)(烧灼)

    • test tube opening, transferring loop

  • Hot air sterilization/Baking(干烤)

    • 160-170℃, 2h

    • Glassware, china, syringes, needles, etc

  • Infrared heat: similar to baking


Moist heat:denaturing proteins and melt lipids

  • Autoclaving(高压蒸汽灭菌)

    • Most commonly used and effective

    • 121℃ (103.4kPa), 15-20min

    • killing both vegetative organisms and

      endospores

  • Boiling

    • 100 ℃ (105℃ with 2% Na2CO2) , 15-20min

    • cidal for vegetative cells but not necessarily spores

  • Regular Steam (Arnold Sterilizer)

    • 100 ℃, 15-20min

    • cidal for vegetative cells but not necessarily spores


  • Pasteurization(巴氏消毒法)

    • to kill pathogens in readily perishable objects (milk, wine)

    • flash method (瞬间法): 71.6℃, 15s

    • holding method (持续法): 62.9℃, 30 min

  • Fractional sterilization(间歇蒸气灭菌法)

    • alternating exposure and cooling time for a consecutive period:

      Steam heating (100℃, 30 min)30℃ for endospores to germinate 100℃, 30 min to kill germinated endospores 30-37℃ overnight for remaining endospores to germinate 100℃, 60 min to kill last remaining germinated endospores

    • for sugar- or milk-containing culture media


Moist Heat vs Dry Heat

Moist heat Dry heat

Penetrating potency higherlower

Temp for protein clotting lowerhigher

Extra heat released yesno

from condensation

Sterilizing potency: Moist heat >> Dry heat


Radiation

  • Ultraviolet (UV) radiation

    • mechanism: blockage of DNA replication by forming thymidine dimmers

    • microbicidal activity of UV depends on:

      • length of exposure

      • wavelength: 200-300 nm, with the best effect of 265-266nm

      • bulb life (4000hr)

    • very poor penetrating power

    • for air or surface disinfection

      (OR, ID ward, labs)

    • causing eye damage, burns

      and mutation in skin cells


  • Ionizing Radiation(电离辐射)

    • X-rays, gamma rays and high-speed electrons

    • generating more energy and penetrating power than UV

    • to sterilize pharmaceuticals, disposable medical supplies (e.g., syringes, gloves, catheters, sutures) and foods

  • Microwave

    • penetrating non-metal materials (glass, plastics, china)


  • Ultrasound

    • more effective for gram-negative bacteria

    • Lack of thoroughness —— survivors remain

  • Filtration

    • sterilize heat- or chemical-sensitive solutions

    • not effective for virus, ricketia, mycoplasma


Seitz filter


  • Desiccation

    • static effect byinhibiting microbial enzymes

    • not effective against endospores

    • mainly for food reservation

  • Low Temperature (-20℃ ~ -70℃)

    • inhibits microbial growth by slowing down microbial metabolism

    • a special form: lyophalization (freeze-drying), used for long-term (years) reservation of bacteria stocks

      • fast freezing + drying

      • protecting agents (glycerol, serum)


Control Microorganisms with Chemical Agents (Disinfectants and Antiseptics)


Antimicrobial modes of action of disinfectants and antiseptics

  • Denaturation of bacterial proteins by disrupting hydrogen and disulfide bonds

    —— phenol (high conc.), alcohol, heavy-metal (high conc.), acids, alkalies, aldehydes)

  • Damage to bacterial membrane (lipids and/or proteins), causing leakage of intracellular molecules

    —— phenol (low conc.), surfactants, dyes

  • Interference of bacterial enzyme and metabolism

    —— oxidants, heavy-metals (low conc.), alkylating agents


  • Phenol and phenol derivatives

    ——altering membrane permeability and denaturing proteins

    • 0.01% - 0.05% Chlorhexidine(洗必泰)—— vaginal wash, OR hand-wash

    • 3% - 5% carbonic acid or 2% Lysol ——floor or surface disinfection


  • Alcohols

    —— denaturing bacterial proteins and membranes

    • 70% - 75% ethyl or isopropyl alcohol(乙醇或异丙醇)—— skin and thermometer disinfection

      • ineffective against endospores and non-enveloped viruses


  • Heavy metals (Hg2+、Ag+)

    —— denaturing proteins and inactivating enzymes

    • 2% mercurochrome(红汞)or 0.1% merthiolate(硫柳汞)—— skin, mucosa and wound

      • bacteriostatic, ineffective against endospores

    • 1% silver nitrate(硝酸银)—— eye drops for newborns to prevent gonococcal ophthalmia(淋菌性眼炎)


  • Oxidants

    —— oxidation, protein precipitation

    • 0.1% potassium permanganate(高锰酸钾)—— skin, fruits/vegetables

    • 3% peroxide(过氧化氢)—— small trauma wound, skin, mucosa

    • 0.2% - 1% peroxyacetic acid(过氧乙酸)—— plastics, glassware

    • 0.2 – 0.5 ppm cholorines —— water and swimming pool


  • Surfactants

    —— damaging bacterial membranes, inactivating enzymes, protein precipitation

    • 0.05-0.1% bromogeramine (新洁尔灭)—— OR hand-wash, skin, surgical instruments


  • Alkylating agent(烷化剂)

    —— alkylating proteins and nucleic acids

    • formalin (formaldehyde) ——surface disinfection, air, surgical instruments

    • glutaric dialdehyde(戊二醛)—— high-precision instruments, endoscopes

    • 50mg/L epoxy ethane(环氧乙烷)——surgical instruments and dressing


  • Dyes

    —— inhibiting bacterial growth by interfering with oxidation

    • 2% - 4% methyl violet(龙胆紫)—— wound disinfection


  • Acids and alkalies

    —— destroying cell membrane and cell wall, denaturing proteins

    • 5-10ml/m3 acetic acid evaporation ——air disinfection

    • quicklime [Ca(OH)2] ——floor and excretion (feces, urine, sputum, pus) disinfection


Effectiveness of antimicrobial agents are affected by ——

  • The concentration/intensity and nature of the disinfectant;

  • Length of exposure;

  • Species and number of the microbe(s);

  • Temperature and humidity;

  • Acidity (pH);

  • Presence of organic substances;

  • Presence of chemical antagonists

  • The nature of the material bearing the microbes


Summary 1. Application of chemical disinfectants

Patient excretionChlorines, 5% carbonic acid, 2% Lysol

Skin (hands)2% Lysol, 0.2-0.4% peroxyacetic acid(过氧乙酸)for HBV, 70% ethyl alcohol, 2% mercurochrome(红汞)

Mucosaoral- 3% peroxide; uri-reproductive- 0.01-0.05% Chlorhexidine(洗必泰), 0.1% potassium permanganate(高锰酸钾); newborn eyes- 1% silver nitrate


Drinking waterChlorines

Toilets, sewagequicklime [Ca(OH)2]

Air (OR, ID ward)formalin steam (12.5-25ml/m3,12-24h),formalin 40ml + potassium permanganate 30g/m3;

HBV ward- peroxyacetic acid 3g/m3 90min

Glassware, china,0.5% iodophores, 0.2-0.4% peroxyacetic

Rubber, metal acid

devices


Summary 2. Potency levels of chemical disinfectants

Potency DefinitionExamples

High Killing all microbes including glutaric dialdehyde(戊二醛),

endospores and TBformaldehyde peroxyacetic acid(过氧乙酸),epoxy ethane(环氧乙烷)

Medium Killing all non-spore microbesalcohol, chlorines, iodophores

including TB

Low Killing vegetative bacteria chlorhexidine(洗必泰),

and lipophilic (enveloped) bromogeramine(新洁尔灭)

viruses, but resisted by

endospores, TB and hydrophilic

(non-enveloped) viruses


Summary 3. Spore-killing effects of chemical disinfectants

Spore-killing disinfectins

  • glutaric dialdehyde(戊二醛), formaldehyde(甲醛), Iodines, H2O2, epoxy ethane(环氧乙烷)

    Non spore-killing disinfectins

  • alcohols, phenols, chlorhexidine(洗必泰),

    bromogeramine(新洁尔灭)


Medical Microbiology

Disinfection and Sterilization

——For the course of Medical Microbiology for MBBS foreign students,Class 2006/2011, SYSU

September 18, 2007

Mengfeng Li (黎孟枫), M.D.

Department of Microbiology, Zhongshan School of Medicine, SYSU, Guangzhou, China

[email protected]


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